This proposal is a continuation of work to describe the nature of the molecular target of ethanol action in nerve cells, and to understand the manner in which nerve cells respond to chronic exposure to ethanol. A line of pheochromocytoma cells, PC12, is used to examine the acute actions of ethanol on membrane channels. It is also used to determine what electrophysiological changes are correlated with changes which have been shown to occur, using biochemical techniques, in the calcium channel population of the cells after chronic ethanol exposure. These studies use patch clamp techniques. Another line of study will examine the relationship between channel structure and ethanol sensitivity, using an oocyte expression system. RNA of known sequence and physiological activity will be injected into the oocyte, and the ethanol sensitivity of the translated product will be examined electrophysiologically. In addition to using a family of mutants of slightly different sequence, site-directed mutagenesis techniques will be used to produce new sequences, designed to test the contribution of various regions of the protein receptor/channel to ethanol sensitivity. The knowledge gained from this project should help to increase our understanding of alcohol intoxication and alcoholism.